Methods of identifying inhibitors of sensor histidine kinases through rational drug design

Abstract

The present invention provides N-terminal truncated transmembrane sensor histidine kinases that retain their ability to be autophophorylated and/or their related histidine kinase activity. The N-terminal truncated transmembrane sensor histidine kinases are useful for obtaining detailed three-dimensional structural data of the catalytic portion of the protein. The three-dimensional structural data is included as part of the invention. In addition, the present invention provides methodology for related structure based rational drug design using the three-dimensional data. Nucleotide and amino acid sequences of the N-terminal truncated transmembrane sensor histidine kinases are also provided.

What is claimed is: 
     
       1. A method of using the three-dimensional structure of an N-terminal truncated transmembrane sensor histidine kinase (N t  TSHK) in a drug screening assay comprising: (a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined from one or more sets of atomic coordinates in FIG. 8 and FIG. 9; wherein said selecting is performed in conjunction with computer modeling;   (b) contacting the potential drug with a first polypeptide comprising a first N t  TSHK; and   (c) detecting the binding of the potential drug with said first polypeptide; wherein a potential drug is selected as a drug if the potential drug binds to said first polypeptide.   
     
     
       2. The method of claim 1 wherein said first polypeptide comprises the N t  TSHK having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 12 having a conservative amino acid substitution. 
     
     
       3. The method of claim 1 wherein said first polypeptide comprises the N t  TSHK having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 having a conservative amino acid substitution. 
     
     
       4. The method of claim 1 wherein said first polypeptide is labeled. 
     
     
       5. The method of claim 1 wherein said first polypeptide is bound to a solid support. 
     
     
       6. The method of claim 1 further comprising: (d) contacting the potential drug with an N t  TSHK for NMR analysis; wherein a binding complex forms between the potential drug and said N t  TSHK for NMR analysis; wherein said N t  TSHK for NMR analysis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 12 having a conservative amino acid substitution, SEQ ID NO: 14, and SEQ ID NO: 14 having a conservative amino acid substitution;   (e) determining the three-dimensional structure of the binding complex by NMR;   (f) selecting a candidate drug by performing structure based rotional drug design with the three-dimensional structure determined for the binding complex; wherein said selecting is performed in conjunction with computer modeling;   (g) contacting the candidate drug with a second polypeptide comprising a second N t  TSHK; and   (h) detecting the binding of the candidate drug with said second polypeptide; wherein a candidate drug is selected as a drug if the candidate drug binds to said second polypeptide; wherein said first N t  TSHK and said second N t  TSHK can be but are not necessarily the same; and wherein said first polypeptide and said second polypeptide can be but are not necessarily the same.   
     
     
       7. The method of claim 6 wherein said second N t  TSHK comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 12 having a conservative amino acid substitution. 
     
     
       8. The method of claim 6 wherein said second N t  TSHK comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 having a conservative amino acid substitution. 
     
     
       9. The method of claim 6 wherein said second polypeptide is labeled. 
     
     
       10. The method of claim 6 wherein said second polypeptide is bound to a solid support. 
     
     
       11. A method of using the three-dimensional structure of an N-terminal truncated transmembrane sensor histidine kinase (N t  TSHK) in a drug screening assay comprising: (a) selecting a potential drug by performing structure based rational drug design with the three-dimensional structure defined by the set of atomic coordinates in FIG. 8; wherein said selecting is performed in conjunction with computer modeling;   (b) contacting the potential drug with a first polypeptide comprising a first N t  TSHK in the presence of a protein histidine kinase; wherein in the absence of the potential drug the protein histidine kinase phosphorylates said first N t  TSHK; and   (c) determining the amount of phosphorylation of said first N t  TSHK; wherein a drug is selected when a decrease in amount of phosphorylation of said first N t  TSHK is determined in the presence of the potential drug relative to in its absence.   
     
     
       12. The method of claim 11 and wherein said N t  TSHK is incapable of phosphorylating a transmembrane sensor histidine kinase (TSHK) but contains a histidine that can be phosphorylated by the TSHK. 
     
     
       13. The method of claim 12 wherein said first N t  TSHK comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 12 having a conservative amino acid substitution. 
     
     
       14. The method of claim 11 further comprising: (d) contacting the potential drug with an N t  TSHK for NMR analysis; wherein a binding complex forms between the potential drug and said N t  TSHK for NMR analysis; wherein said N t  TSHK for NMR analysis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 12 having a conservative amino acid substitution;   (e) determining the three-dimensional structure of the binding complex by NMR;   (f) selecting a candidate drug by performing structural based rational drug design with the three-dimensional structure determined for the binding complex; wherein said selecting is performed in conjunction with computer modeling;   (g) contacting the candidate drug with a second polypeptide comprising a second N t  TSHK in the presence of a protein histidine kinase; wherein in the absence of the candidate drug the protein histidine kinase phosphorylates said second N t  TSHK; and   (h) determining the amount of phosphorylation of said second N t  TSHK; wherein a drug is selected when a decrease in the amount of phosphorylation of said second N t  TSHK is determined in the presence of the candidate drug relative to in its absence; wherein said first N t  TSHK and said second N t  TSHK can be but are not necessarily the same; and wherein said first polypeptide and said second polypeptide can be but are not necessarily the same.   
     
     
       15. A method of using the three-dimensional structure of an N-terminal truncated transmembrane sensor histidine kinase (N t  TSHK) in a drug screening assay comprising: (a) selecting a potential drug by performing structural based rotational drug design with the three-dimensional structure determined defined by the set of atomic coordinates in FIG. 9; wherein said selecting is performed in conjunction with computer modeling;   (b) contacting the potential drug with a first polypeptide comprising a first N t  TSHK in the presence of a protein histidine substrate; wherein in the absence of the potential drug the protein histidine kinase substrate is phosphorylated by said first N t  TSHK; and   (c) determining the amount of phosphorylation of the protein histidine kinase substrate; wherein a drug is selected when a decrease in the amount of phosphorylation of the protein histidine kinase substrate is determined in the presence of the potential drug relative to in its absence.   
     
     
       16. The method of claim 15 wherein said N t  TSHK is capable of phosphorylating a transmembrane sensor histidine kinase (TSHK) but lacks the autophosphorylatable histidine. 
     
     
       17. The method of claim 16 wherein said first N t  TSHK has amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14 having a conservative amino acid substitution. 
     
     
       18. The method of claim 15 further comprising: (d) contacting the potential drug with an N t  TSHK for NMR analysis; wherein a binding complex forms between the potential drug and said N t  TSHK for NMR analysis; wherein said N t  TSHK for NMR analysis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 14 having a conservative amino acid substitution;   (e) determining the three-dimensional structure of the binding complex by NMR; and   (f) selecting a candidate drug by performing structural based rational drug design with the three-dimensional structure determined for the binding complex; wherein said selecting is performed in conjunction with computer modeling;   (g) contacting the candidate drug with a second polypeptide comprising a second N t  TSHK in the presence of a protein histidine kinase substrate; wherein in the absence of the candidate drug the protein histidine kinase substrate is phosphorylated by said second N t  TSHK; and   (h) determining the amount of phosphorylation of the protein histidine kinase substrate; wherein a drug is selected when a decrease in the amount of phosphorylation of the protein histidine kinase substrate is determined in the presence of the candidate drug relative to in its absence; wherein said first N t  TSHK and said second N t  TSHK can be but are not necessarily the same; and wherein said first polypeptide and said second polypeptide can be but are not necessarily the same.